Evaluation of SARS-CoV-2 neutralizing antibodies using a CPE-based colorimetric live virus micro-neutralization assay in human serum samples.

The micro-neutralization assay is a fundamental test in virology, immunology, vaccine assessment, and epidemiology studies. Since the SARS-CoV-2 outbreak at the end of December 2019 in China, it has become extremely important to have well-established and validated diagnostic and serological assays for this new emerging virus. Here, we present a micro-neutralization assay with the use of SARS-CoV-2 wild type virus with two different methods of read-out. We evaluated the performance of this assay using human serum samples taken from an Italian seroepidemiological study being performed at the University of Siena, along with the human monoclonal antibody CR3022 and some iper-immune animal serum samples against Influenza and Adenovirus strains. The same panel of human samples have been previously tested in enzyme-linked immunosorbent assay (ELISA) as a pre-screening. Positive, borderline, and negative ELISA samples were evaluated in neutralization assay using two different methods of read-out: subjective (by means of an inverted optical microscope) and objective (by means of a spectrophotometer). Our findings suggest that at least 50% of positive ELISA samples are positive in neutralization as well, and that method is able to quantify different antibody concentrations in a specific manner. Taken together, our results confirm that the colorimetric cytopathic effect-based microneutralization assay could be used as a valid clinical test method for epidemiological and vaccine studies.

Serological Evidence for Circulation of Influenza D Virus in the Ovine Population in Italy.

Influenza D virus (IDV) is a novel orthomyxovirus initially isolated from pigs exhibiting influenza-like disease in the USA. Since then, IDV has been detected worldwide in several host species, including livestock animals, whilst specific antibodies have been identified in humans, raising concerns about interspecies transmission and zoonotic risks. Few data regarding the seroprevalence of IDV in small ruminants have been available to date. In this study, we assessed the prevalence of antibodies against IDV in ovine serum samples in Sicily, Southern Italy. Six hundred serum samples, collected from dairy sheep herds located in Sicily in 2022, were tested by haemagglutination inhibition (HI) and virus neutralization (VN) assays using reference strains, D/660 and D/OK, representative of two distinct IDV lineages circulating in Italy. Out of 600 tested samples, 168 (28.0%) tested positive to either IDV strain D/660 or D/OK or to both by HI whilst 378 (63.0%) tested positive to either IDV strain D/660 or D/OK or to both by VN. Overall, our findings demonstrate that IDV circulates in ovine dairy herds in Sicily. Since IDV seems to have a broad host range and it has zoonotic potential, it is important to collect epidemiological information on susceptible species.

Haemagglutination inhibition and virus microneutralisation serology assays: use of harmonised protocols and biological standards in seasonal influenza serology testing and their impact on inter-laboratory variation and assay correlation: A FLUCOP collaborative study.

Introduction: The haemagglutination inhibition assay (HAI) and the virus microneutralisation assay (MN) are long-established methods for quantifying antibodies against influenza viruses. Despite their widespread use, both assays require standardisation to improve inter-laboratory agreement in testing. The FLUCOP consortium aims to develop a toolbox of standardised serology assays for seasonal influenza. Building upon previous collaborative studies to harmonise the HAI, in this study the FLUCOP consortium carried out a head-to-head comparison of harmonised HAI and MN protocols to better understand the relationship between HAI and MN titres, and the impact of assay harmonisation and standardisation on inter-laboratory variability and agreement between these methods. Methods: In this paper, we present two large international collaborative studies testing harmonised HAI and MN protocols across 10 participating laboratories. In the first, we expanded on previously published work, carrying out HAI testing using egg and cell isolated and propagated wild-type (WT) viruses in addition to high-growth reassortants typically used influenza vaccines strains using HAI. In the second we tested two MN protocols: an overnight ELISA-based format and a 3-5 day format, using reassortant viruses and a WT H3N2 cell isolated virus. As serum panels tested in both studies included many overlapping samples, we were able to look at the correlation of HAI and MN titres across different methods and for different influenza subtypes. Results: We showed that the overnight ELISA and 3-5 day MN formats are not comparable, with titre ratios varying across the dynamic range of the assay. However, the ELISA MN and HAI are comparable, and a conversion factor could possibly be calculated. In both studies, the impact of normalising using a study standard was investigated, and we showed that for almost every strain and assay format tested, normalisation significantly reduced inter-laboratory variation, supporting the continued development of antibody standards for seasonal influenza viruses. Normalisation had no impact on the correlation between overnight ELISA and 3-5 day MN formats.

An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study.

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.

Correlation of Influenza B Haemagglutination Inhibiton, Single-Radial Haemolysis and Pseudotype-Based Microneutralisation Assays for Immunogenicity Testing of Seasonal Vaccines.

Influenza B is responsible for a significant proportion of the global morbidity, mortality and economic loss caused by influenza-related disease. Two antigenically distinct lineages co-circulate worldwide, often resulting in mismatches in vaccine coverage when vaccine predictions fail. There are currently operational issues with gold standard serological assays for influenza B, such as lack of sensitivity and requirement for specific antigen treatment. This study encompasses the gold standard assays with the more recent Pseudotype-based Microneutralisation assay in order to study comparative serological outcomes. Haemagglutination Inhibition, Single Radial Haemolysis and Pseudotype-based Microneutralisation correlated strongly for strains in the Yamagata lineage; however, it correlated with neither gold standard assays for the Victoria lineage.

Characterization of antibody response in asymptomatic and symptomatic SARS-CoV-2 infection.

SARS-CoV-2 pandemic is causing high morbidity and mortality burden worldwide with unprecedented strain on health care systems. To investigate the time course of the antibody response in relation to the outcome we performed a study in hospitalized COVID-19 patients. As comparison we also investigated the time course of the antibody response in SARS-CoV-2 asymptomatic subjects. Study results show that patients produce a strong antibody response to SARS-CoV-2 with high correlation between different viral antigens (spike protein and nucleoprotein) and among antibody classes (IgA, IgG, and IgM and neutralizing antibodies). The antibody peak is reached by 3 weeks from hospital admission followed by a sharp decrease. No difference was observed in any parameter of the antibody classes, including neutralizing antibodies, between subjects who recovered or with fatal outcome. Only few asymptomatic subjects developed antibodies at detectable levels.

Evaluation of Varicella Immunity during Pregnancy in Apulia Region, Southern Italy.

Varicella is a highly contagious, infectious disease caused by the varicella-zoster virus. Those at higher risk of severe complications are immunocompromised individuals, adults, non-immune pregnant women, and newborns. According to the gestational time, when varicella-zoster virus infection is acquired during pregnancy, serious complications can potentially occur for both the woman and the fetus. The present study was conducted to assess the profile of varicella susceptibility in pregnant women in Apulia, a large region in Southern Italy, from 2016 to 2019. The data showed that pregnant women between the age of 15-24 and 40-49 years, the youngest and the oldest, respectively, are the most protected against varicella-zoster virus infection, exceeding the prevalence rate of 90%. Conversely, pregnant women between the age of 25 and 34 years seem to be the most vulnerable and the most at risk for acquiring varicella-zoster virus infection during pregnancy. Analysis of the immunity status against varicella should be introduced as a screening test before pregnancy, together with a strategic vaccination campaign targeting non-immune women of childbearing age, in order to reduce the risk of congenital and perinatal varicella.

Seroprevalence of Hepatitis E Virus in Forestry Workers from Trentino-Alto Adige Region (Northern Italy).

People with some occupational or recreational activities, such as hunters and veterinarians, may have increased risk to be infected by the hepatitis E virus (HEV). The aim of the present study was to establish whether forestry workers could be considered at a higher risk of HEV infection than a control group. One hundred and fifty sera from forestry workers and a control group of 85 sera were analysed by anti-HEV IgG antibodies detection using a commercial ELISA kit. The anti-HEV IgG seroprevalence was 14% for forestry workers and 9.4% for the control group. Comparing the risk of HEV infection in the two groups, there was no difference in the odds ratio. However, the seroprevalence in older subjects was higher in the forestry workers than in the control group. Two sera from forestry workers were also positive for anti-HEV IgM, and, in one of them, HEV-RNA was detected. Our findings showed an increase of seroprevalence with age, which is likely to reflect cumulative exposure to HEV over time. The occupation of forestry workers did not seem to be associated with a higher risk of HEV infection. The study provided new insights into the risk of acquiring HEV in occupational exposure workers with open-air activities.

Influenza D Virus: Serological Evidence in the Italian Population from 2005 to 2017.

Influenza D virus is a novel influenza virus, which was first isolated from an ailing swine in 2011 and later detected in cattle, suggesting that these animals may be a primary natural reservoir. To date, few studies have been performed on human samples and there is no conclusive evidence on the ability of the virus to infect humans. The aim of this serological study was to assess the prevalence of antibodies against influenza D virus in human serum samples collected in Italy from 2005 to 2017. Serum samples were analysed by haemagglutination inhibition and virus neutralization assays. The results showed that the prevalence of antibodies against the virus increased in the human population in Italy from 2005 to 2017, with a trend characterized by a sharp increase in some years, followed by a decline in subsequent years. The virus showed the ability to infect and elicit an immune response in humans. However, prevalence peaks in humans appear to follow epidemics in animals and not to persist in the human population.

Seroprevalence study of Toscana virus and viruses belonging to the Sandfly fever Naples antigenic complex in central and southern Italy.

Sandfly fever viruses are transmitted by the bite of phlebotomine sandflies; serotypes sandfly fever Naples virus, sandfly fever Sicilian virus and sandfly fever Cyprus virus cause febrile illness, whereas Toscana virus (TOSV) may cause neuroinvasive infections. Although TOSV is an important cause of aseptic meningitis in central and southern Italy, in many cases the infection is asymptomatic, leading to underestimation of the actual spread of the virus. This serosurvey aimed to assess the seroprevalence of TOSV in a random population in Siena (Tuscany, central Italy) in 2003-2004 and 2013-2014 and Bari (Apulia, southern Italy) in 2004 and 2015. 2132 serum samples were tested for the presence of anti-TOSV/SFNV IgG by means of ELISA and IFA commercial tests. Seroprevalence rates were compared in the two cities and over a ten-year period in the same city. Seroprevalence results in the Siena population (22.95% in 2003-2004 vs 26.75% in 2013-2014) confirmed the endemic circulation of TOSV and closely related viruses in central Italy, without major changes over the last decade, while no significant prevalence was observed in Bari (2.90% in 2004 vs 1.85% in 2015).